Anti-annexin a2 monoclonal antibodies

ABSTRACT

The present invention relates to an antigen-binding protein, preferably a monoclonal antibody, against annexin A2 (ANXA2), comprising (i) a heavy chain variable domain comprising a VHCDR1 of sequence GYSITSGYSWH, a VHCDR2 of sequence YIHYSGSTKYNPSLKS and a VHCDR3 of sequence GSNYGFDY; and (ii) a light chain variable domain comprising a VLCDR1 of sequence KSSQSLLYSNDQKNYLA, a VLCDR2 of sequence WASIRES, and a VLCDR3 of sequence QQYYIYPLT. Also provided is an ANXA2 binding protein comprising (i) a heavy chain variable domain comprising a VHCDR1 of sequence VYSITSGYSWH; a VHCDR2 of sequence YIHYSGSTKYNPSLKS, and a VHCDR3 of sequence GTDNAVDY; and (ii) a light chain variable domain comprising a VLCDR1 of sequence KSSQSLLYSSNQKNYLA, a VLCDR2 of sequence WASSRES, and a VLCDR3 of sequence QQYYIYPLT. The antibodies preferably bind to an N-linked glycan on ANXA2, and are internalised upon binding. They may be conjugated with cytotoxins and may be used in the treatment or detection of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of Singapore applicationNo. 10201606178X, filed 26 Jul. 2016, the contents of it being herebyincorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates generally to antibodies. Specifically, theinvention relates to anti-annexin A2 monoclonal antibodies and usesthereof.

BACKGROUND OF THE INVENTION

Annexin A2 (ANXA2) is involved in diverse cellular processes andclinical associations, especially in cancer progression. It is acalcium-dependent phospholipid-binding protein whose function is to helporganize exocytosis of intracellular proteins to the extracellulardomain. ANXA2 is a pleiotropic protein meaning that its function isdependent on place and time in the body.

Increased expression of ANXA2 is frequently observed in a broad spectrumof cancer cells. ANXA2 is overexpressed in acute lymphoblastic leukemia(ALL), APL, breast cancer, colorectal carcinoma (CRC), gastric cancer,glioma, hepatocellular carcinoma (HCC), lung cancer, multiple myeloma(MM), oral squamous cell carcinoma (OSCC), and pancreatic cancer. Theup-regulation of ANXA2 in cancer may have several clinical applications,including as a diagnostic marker for early detection, a predictivefactor for prognosis, or a marker for drug resistance.

To date there are no anti-ANXA2 monoclonal antibodies that could be usedfor antibody therapy or as antibody-drug conjugates for cancertreatment. In addition, there are no reported antibodies targetingunique cancer specific glycoforms of ANXA2. There is therefore a need todevelop novel antibodies against ANXA2 that address the disadvantages ofthe antibodies that are currently available.

SUMMARY

In one aspect, there is provided an antigen-binding protein, or anantigen-binding fragment thereof, comprising (i) a heavy chain variabledomain comprising a VHCDR1 having the amino acid sequence GYSITSGYSWH(SEQ ID NO: 9); a VHCDR2 having the amino acid sequence YIHYSGSTKYNPSLKS(SEQ ID NO: 10) and a VHCDR3 having the amino acid sequence GSNYGFDY(SEQ ID NO: 11); and (ii) a light chain variable domain comprising aVLCDR1 having the amino acid sequence KSSQSLLYSNDQKNYLA (SEQ ID NO: 12),a VLCDR2 having the amino acid sequence WASIRES (SEQ ID NO: 13), and aVLCDR3 having the amino acid sequence QQYYIYPLT (SEQ ID NO: 14).

In another aspect, there is provided an antigen-binding protein, or anantigen-binding fragment thereof, comprising (i) a heavy chain variabledomain comprising a VHCDR1 having the nucleic acid sequenceGCTACTCCATCACCAGTGGTTATAGCTGGCAC (SEQ ID NO: 15); a VHCDR2 having thenucleic acid sequence CATACACTACAGTGGTAGCACTAAGTACAACCCATCTCTCAAAAGTC(SEQ ID NO: 16), and a VHCDR3 having the nucleic acid sequenceGGAGTAACTACGGATTTGACTACT (SEQ ID NO: 17); and (ii) a light chainvariable domain comprising a VLCDR1 having the nucleic acid sequenceAGTCCAGTCAGAGCCTTTTATATAGTAACGATCAAAAGAACTACTTGGCCT (SEQ ID NO: 18), aVLCDR2 having the nucleic acid sequence GGGCATCTATTAGGGAATCTG (SEQ IDNO: 19), and a VLCDR3 having the nucleic acid sequenceAGCAATATTATATCTATCCTCTCACGT (SEQ ID NO: 20).

In another aspect, there is provided an antigen-binding protein, or anantigen-binding fragment thereof, comprising (i) a heavy chain variabledomain comprising a VHCDR1 having the amino acid sequence VYSITSGYSWH(SEQ ID NO: 21); a VHCDR2 having the amino acid sequenceYIHYSGSTKYNPSLKS (SEQ ID NO: 10), and a VHCDR3 having the amino acidsequence GTDNAVDY (SEQ ID NO: 22); and (ii) a light chain variabledomain comprising a VLCDR1 having the amino acid sequenceKSSQSLLYSSNQKNYLA (SEQ ID NO: 23), a VLCDR2 having the amino acidsequence WASSRES (SEQ ID NO: 24), and a VLCDR3 having the amino acidsequence QQYYIYPLT (SEQ ID NO: 14).

In another aspect, there is provided, an antigen-binding protein, or anantigen-binding fragment thereof, comprising (i) a heavy chain variabledomain comprising a VHCDR1 having the nucleic acid sequenceTCTACTCCATCACCAGTGGTTATAGCTGGCACT (SEQ ID N: 25); a VHCDR2 having thenucleic acid sequence ACATACACTACAGTGGTAGTACTAAGTACAACCCATCTCTCAAAAGTC(SEQ ID NO: 26), and a VHCDR3 having the nucleic acid sequenceGGACCGACAATGCTGTGGACTACT (SEQ ID NO: 27); and (ii) a light chainvariable domain comprising a VLCDR1 having the nucleic acid sequenceAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCT SEQ ID NO: 28), aVLCDR2 having the nucleic acid sequence GGGCATCCAGTAGGGAATCTG (SEQ IDNO: 29), and a VLCDR3 having the nucleic acid sequenceAGCAATATTATATCTATCCTCTCACGT (SEQ ID NO: 20).

In another aspect, there is provided a composition comprising aphysiologically acceptable carrier and a therapeutically effectiveamount of the antigen-binding protein, or an antigen-binding fragmentthereof, as described herein.

In another aspect, there is provided use of an antigen-binding protein,or an antigen-binding fragment thereof, as described herein, in themanufacture of a medicament for treating or preventing cancer.

In another aspect, there is provided a method for detecting cancer in asubject, the method comprising: contacting a sample obtained from thesubject with an antigen-binding protein, or an antigen-binding fragmentthereof as described herein in vitro; detecting the binding of theantigen-binding protein, or an antigen-binding fragment thereof in thesample; correlating the binding with a level of binding in a controlsample to determine the level of binding in the sample, wherein anincrease in the level of binding in the sample relative to the controlsample is indicative of cancer.

In another aspect, there is provided a method for identifying a subjectsusceptible to cancer the method comprising: contacting a sampleobtained from the subject with an antigen-binding protein, or anantigen-binding fragment thereof as described herein in vitro; detectingthe binding of the antigen-binding protein, or an antigen-bindingfragment thereof in the sample; correlating the binding with a level ofbinding in a control sample to determine the level of binding in thesample, wherein an increase in the level of binding in the samplerelative to the control sample indicates that the subject is susceptibleto cancer.

In another aspect, there is provided a kit when used in the method asdescribed herein, comprising an antigen-binding protein, orantigen-binding fragment thereof as described herein, together withinstructions for use.

DEFINITIONS

The following are some definitions that may be helpful in understandingthe description of the present invention. These are intended as generaldefinitions and should in no way limit the scope of the presentinvention to those terms alone, but are put forth for a betterunderstanding of the following description.

The term “antigen binding protein” as used herein refers to antibodies,antibody fragments and other protein constructs, such as domains, whichare capable of binding to ANXA2.

The term “antibody” is used herein in the broadest sense to refer tomolecules with an immunoglobulin-like domain and includes monoclonal,recombinant, polyclonal, chimeric, humanised, bispecific andheteroconjugate antibodies; a chimeric antigen receptor (CAR), a singlevariable domain, a domain antibody, antigen binding fragments,immunologically effective fragments, single chain Fv, diabodies,Tandabs™, etc (for a summary of alternative “antibody” formats seeHolliger and Hudson, Nature Biotechnology, 2005, Vol 23, No. 9,1126-1136).

A “chimeric antigen receptor” may comprise an extracellular domaincomprising the antigen binding domain, a transmembrane domain and anintracellular signalling domain. The extracellular domain may be linkedto the transmembrane domain by a linker. The extracellular domain mayalso comprise a signal peptide.

The phrase “single variable domain” refers to an antigen binding proteinvariable domain (for example, V_(H), V_(HH), V_(L)) that specificallybinds an antigen or epitope independently of a different variable regionor domain.

A “domain antibody” or “dAb” may be considered the same as a “singlevariable domain” which is capable of binding to an antigen. A singlevariable domain may be a human antibody variable domain, but alsoincludes single antibody variable domains from other species such asrodent (for example, as disclosed in WO 00/29004), nurse shark andCamelid V_(HH) dAbs. Camelid V_(HH) are immunoglobulin single variabledomain polypeptides that are derived from species including camel,llama, alpaca, dromedary, and guanaco, which produce heavy chainantibodies naturally devoid of light chains. Such V_(HH) domains may behumanised according to standard techniques available in the art, andsuch domains are considered to be “domain antibodies”. As used hereinV_(H) includes camelid V_(HH) domains.

As used herein the term “domain” refers to a folded protein structurewhich has tertiary structure independent of the rest of the protein.Generally, domains are responsible for discrete functional properties ofproteins, and in many cases may be added, removed or transferred toother proteins without loss of function of the remainder of the proteinand/or of the domain. A “single variable domain” is a folded polypeptidedomain comprising sequences characteristic of antibody variable domains.It therefore includes complete antibody variable domains and modifiedvariable domains, for example, in which one or more loops have beenreplaced by sequences which are not characteristic of antibody variabledomains, or antibody variable domains which have been truncated orcomprise N- or C-terminal extensions, as well as folded fragments ofvariable domains which retain at least the binding activity andspecificity of the full-length domain. A domain can bind an antigen orepitope independently of a different variable region or domain.

An antigen binding fragment may be provided by means of arrangement ofone or more CDRs on non-antibody protein scaffolds such as a domain. Thedomain may be a domain antibody or may be a domain which is a derivativeof a scaffold selected from the group consisting of CTLA-4, lipocalin,SpA, an Affibody, an avimer, GroE1, transferrin, GroES andfibronectin/adnectin, which has been subjected to protein engineering inorder to obtain binding to an antigen, such as ANXA2, other than thenatural ligand.

An antigen binding fragment or an immunologically effective fragment maycomprise partial heavy or light chain variable sequences. Fragments areat least 5, 6, 8 or 10 amino acids in length. Alternatively thefragments are at least 15, at least 20, at least 50, at least 75, or atleast 100 amino acids in length.

The term “specifically binds” as used throughout the presentspecification in relation to antigen binding proteins means that theantigen binding protein binds to ANXA2 with no or insignificant bindingto other (for example, unrelated) proteins. However, the term does notexclude the fact that the antigen binding proteins may also becross-reactive with closely related molecules. The antigen bindingproteins described herein may bind to ANXA2 with at least 2, 5, 10, 50,100, or 1000 fold greater affinity than they bind to closely relatedmolecules.

The term “neutralises” as used throughout the present specificationmeans that the biological activity of ANXA2 is reduced in the presenceof an antigen binding protein as described herein in comparison to theactivity of ANXA2 in the absence of the antigen binding protein, invitro or in vivo. Neutralisation may be due to one or more of blockingANXA2 binding to its receptor, preventing ANXA2 from activating itsreceptor, down regulating ANXA2 or its receptor, or affecting effectorfunctionality. The reduction or inhibition in biological activity may bepartial or total. A neutralising antigen binding protein may neutralisethe activity of ANXA2 by at least 20%, 30% 40%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or100% relative to ANXA2 activity in the absence of the antigen bindingprotein. Neutralisation may be determined or measured using one or moreassays known to the skilled person or as described herein. For example,antigen binding protein binding to ANXA2 can be assessed in a sandwichELISA, by BIAcore™, FMAT, FORTEbio, or similar in vitro assays.

“CDRs” are defined as the complementarity determining region amino acidsequences of an antigen binding protein. These are the hypervariableregions of immunoglobulin heavy and light chains. There are three heavychain and three light chain CDRs (or CDR regions) in the variableportion of an immunoglobulin. Thus, “CDRs” as used herein refers to allthree heavy chain CDRs, all three light chain CDRs, all heavy and lightchain CDRs, or at least two CDRs.

As used herein, the term “promoter” is intended to refer to a region ofDNA that initiates transcription of a particular gene.

As used herein, the term “cancerous” relates to being affected by orshowing abnormalities characteristic of cancer.

As used herein, the term “biological sample” or “sample” is meant asample of tissue or cells from a patient that has been obtained from,removed or isolated from the patient.

The term “obtained or derived from” as used herein is meant to be usedinclusively. That is, it is intended to encompass any nucleotidesequence directly isolated from a biological sample or any nucleotidesequence derived from the sample.

The method as described herein is suitable for use in a sample of freshtissue, frozen tissue, paraffin-preserved tissue and/or ethanolpreserved tissue. The sample may be a biological sample. Non-limitingexamples of biological samples include whole blood or a componentthereof (e.g. plasma, serum), urine, saliva lymph, bile fluid, sputum,tears, cerebrospinal fluid, bronchioalveolar lavage fluid, synovialfluid, semen, ascitic tumor fluid, breast milk and pus. In oneembodiment, the sample of nucleic acid is obtained from blood, amnioticfluid or a buccal smear. In a preferred embodiment, the sample is awhole blood sample.

A biological sample as contemplated herein includes cultured biologicalmaterials, including a sample derived from cultured cells, such asculture medium collected from cultured cells or a cell pellet.Accordingly, a biological sample may refer to a lysate, homogenate orextract prepared from a whole organism or a subset of its tissues, cellsor component parts, or a fraction or portion thereof. A biologicalsample may also be modified prior to use, for example, by purificationof one or more components, dilution, and/or centrifugation.

As used herein, the term “detectable label” or “reporter” refers to adetectable marker or reporter molecules, which can be attached tonucleic acids. Typical labels include fluorophores, radioactiveisotopes, ligands, chemiluminescent agents, metal sols and colloids, andenzymes. Methods for labeling and guidance in the choice of labelsuseful for various purposes are discussed, e.g., in Sambrook et al., inMolecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989) and Ausubel et al., in Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley-Intersciences (1987).

As used herein, the term “about”, in the context of concentrations ofcomponents of the formulations, typically means +/−5% of the statedvalue, more typically +/−4% of the stated value, more typically +/−3% ofthe stated value, more typically, +/−2% of the stated value, even moretypically +/−1% of the stated value, and even more typically +/− 0.5% ofthe stated value.

Throughout this disclosure, certain embodiments may be disclosed in arange format. It should be understood that the description in rangeformat is merely for convenience and brevity and should not be construedas an inflexible limitation on the scope of the disclosed ranges.Accordingly, the description of a range should be considered to havespecifically disclosed all the possible sub-ranges as well as individualnumerical values within that range. For example, description of a rangesuch as from 1 to 6 should be considered to have specifically disclosedsub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6 etc., as well as individual numbers within thatrange, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of thebreadth of the range.

Certain embodiments may also be described broadly and genericallyherein. Each of the narrower species and subgeneric groupings fallingwithin the generic disclosure also form part of the disclosure. Thisincludes the generic description of the embodiments with a proviso ornegative limitation removing any subject matter from the genus,regardless of whether or not the excised material is specificallyrecited herein.

Unless the context requires otherwise or specifically stated to thecontrary, integers, steps, or elements of the invention recited hereinas singular integers, steps or elements clearly encompass both singularand plural forms of the recited integers, steps or elements.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The invention illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims and non-limitingexamples. In addition, where features or aspects of the invention aredescribed in terms of Markush groups, those skilled in the art willrecognize that the invention is also thereby described in terms of anyindividual member or subgroup of members of the Markush group.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detaileddescription when considered in conjunction with the non-limitingexamples and the accompanying drawings, in which

FIG. 1: Antibody heavy and light chain sequences and isotype of mAb C51.(A) Nucleotide sequence mAb C51. (B) Amino acid sequence of mAb C51. (C)Isotype of mAb C51 as identified from supernatant of mouse hybridomaculture expressing mAb C51 using the IsoStrip Mouse Monoclonal AntibodyIsotyping Kit (Roche). Antibody Complementarity-Determining Regions areunderlined.

FIG. 2: Reactivity of mAb C51 to Various Types of Cancer and Normal CellLines. (A) Heat map of C51 binding to various cancer cell lines.Reactivity was graded 0-4 based on percentage of mAb C51 binding tovarious cell populations via flow cytometry. (B) Table of C51 binding toovarian and breast cancer cell lines according to a classification basedon the Epithelial-Mesenchymal Transition (EMT).

FIG. 3: Validation of ANXA2 as the antigen target of mAb C51. (A)Immunoprecipitation (IP) with mAb C51 and a commercially availableα-ANXA2 mAb were cross-probed using Western Blot analysis. Similarantigen bands appeared around 39 kDA. (B) ANXA2 siRNA was used toknockdown ANXA2 in IGROV1 cells. Western Blot analysis was performed toevaluate loss of mAb C51 binding to ANXA2-knockdown cell lysate.

FIG. 4: Binding of mAb C51 to an N-glycan epitope. (A) Treatment of themembrane fraction of MCF7 cells with PNGase-F showed loss of C51-bindingto the 39-kDa band antigen band. (B) When cells were treated withtunicamycin and lysed. Western blot analysis showed a loss of the targetantigen for C51.

FIG. 5: Direct cytotoxicity of mAb 51 via oncosis. Increase in propidiumiodide uptake by a range of tumor cell lines when suspended in thepresence of mAb C51 (10 ug/ml).

FIG. 6: Targeted delivery of a toxic payload by mAb C51. The ability ofmAb C51 to deliver the ribosome inactivating protein, saporin intovarious cancer cell lines was assessed. Various cell lines were culturedin the presence of mAb C51 preincubated with a saporin-linked antimouseIgG for 72 hours and assessed via CellTiter-Glo® Assay (Promega).Significant anti-proliferative effects were observed in IGROV1, OVCAR3,SKOV3(D10) and MCF7 which are positive for C51 antigen target comparedto the negative cell lines IOSE523 and BT549.

FIG. 7: Complement-Dependent Cytotoxicity (CDC) of mAb C51. The abilityof mAb C51 to induce CDC was observed in a dose-dependent manner with upto 30% lysis. IGROV1 cells were incubated with increasing concentrationsof mAb C51 in the presence of normal human serum for 2 hours.Cytotoxicity was assessed via LDH release by the CytoTox 96® assay(Promega).

FIG. 8: Targeted delivery of a toxic payload by mAb chC51. The abilityof mAb chC51 to deliver the ribosome inactivating protein, saporin intovarious cancer cell lines was assessed. Various cell lines were culturedin the presence of mAb chC51 preincubated with a saporin-linkedantimouse IgG for 72 hours and assessed via CellTiter-Glo® Assay(Promega). Significant anti-proliferative effects were observed inIGROV1, SKOV3-LUC-D3, MCF7 and HCC1937 cell lines.

FIG. 9: ADCC activity of C51 vs chC51. chC51 but not C51 exhibited ADCCactivity when cultured with MCF7 breast cancer cells. ADCC activity wasmeasured within 6 hour as fold induction of the NFAT ADCC pathway usingan ADCC reporter biossay (Promega).

FIG. 10: Antibody heavy and light chain sequences and isotype of mAb2448. (A) Nucleotide sequence mAb 2448. (B) Amino acid sequence of mAb2448. Antibody Complementarity-Determining Regions are underlined (C)Isotype of mAb 2448 as identified from supernatant of mouse hybridomaculture expressing mAb 2448 using the IsoStrip Mouse Monoclonal AntibodyIsotyping Kit (Roche).

FIG. 11: Reactivity of mAb 2448 to Various Types of Cancer and NormalCell Lines.(A) Heat map of mAb 2448 binding to various cancer celllines. Reactivity was graded 0-4 based on percentage of mAb 2448 bindingto various cell populations via flow cytometry. (B) Table of mAb 2448binding to ovarian and breast cancer cell lines according to aclassification based on the Epithelial-Mesenchymal Transition (EMT). (C)Isogenic breast cancer cell lines MCF7-D10 and MCF7-2101 displayedepithelial and mesenchymal phenotypes of EMT, respectively.Significantly higher levels of the epithelial marker E-Cadherin andlower levels of the mesenchymal marker Vimentin were expressed in MCF7cells versus MCF72101 cells, respectively. Phase contrast microscopyimages revealed EMT-like morphological changes. MCF7-D10 cells were moreepithelial with cuboidal or “cobblestone-like” cells compared toMCF7-2101 cells which were more isolated and elongated. Membranousbinding of 2448 was observed on MCF7-D10 cells but not on the isogenicMCF7-2101 cells by immunocytochemistry. mAb 2448 demonstrated binding(shaded histogram) on live MCF7-D10 cells, but not on MCF7-2101 cells byflow cytometry.

FIG. 12: Validation of ANXA2 as the antigen target of mAb 2448. (A)Immunoprecipitation from the membrane fraction of cell lysate (MF) usingmAb 2448 coupled Protein G beads was identified by Western Blotanalysis. (B) The corresponding band (↑) on a silver-stained gel wasexcised and sent for identification using liquid chromatographytandem-mass spectrometry (LC/MS-MS). After a protein database searchusing MS data, the potential target of ANXA2 was identified. (C) Peptidematches (boxed) from multiple rounds of mass spectrometry spanned acrossthe peptide sequence of ANXA2 (SEQ ID NO: 30).

FIG. 13: Antigen target identification of 2448. (A) Western blotanalysis of antigen immunoprecipitated from human embryonic stem cellsand the membrane fraction of T47D breast cancer cell lines using mAb2448 coupled Protein G beads. (B) The corresponding band on a silverstained gel was excised and identified as ANXA2using mass spectrometryanalysis.

FIG. 14: Validation of ANXA2 as the antigen target of 2448. (A)Immunoprecipitation (IP) using a commercially available α-annexin 2(ANXA2) mAb and mAb 2448 were cross-probed using Western Blot analysis.Similar antigen bands appeared around 39 kDA. (B) ANXA2 siRNA was usedto knockdown ANXA2 in IGROV1 cells. Western Blot analysis was performedto evaluate loss of mAb 2448 binding to ANXA2-knockdown cells lysate.

FIG. 15: Binding of 2448 to the cell surface. mAb 2448 bound on the cellsurface of live IGROV1 cells via flow cytometry. Commercial α-ANXA2antibodies such as those reported in literatures were unable to bind tolive cells. Solid line is negative control; dashed line is mAb 2448.

FIG. 16: Binding of mAb 2448 to an N-glycan epitope. (A) Binding of mAb2448 was sensitive to periodate treatment of Western Blot membranes runwith IGROV1 lysate. (B) Treatment of the membrane fraction of cells withPNGase-F showed loss of 2448-binding to the 39-kDa band antigen band.

FIG. 17: Rapid cellular internalization of mAb 2448. mAb 2448 waslabeled with two pH sensitive dyes, (A) CypHER5E and (B) pH-RODO, thatincreased in fluorescence intensity in low pH environments.Dye-conjugated mAb 2448 internalizes into endosomal and lysosomalcompartments as measured via flow cytometry analysis by an increase intotal mean fluorescence.

FIG. 18: ADC activity of mAb 2448, ch2448 and ch2448ATS. Cytotoxicity ofantibodies (2448 and ch2448) in complex with secondary saporinconjugates (ZAP) was evaluated on ovarian and breast cancer cells.Antibody 2448 and ch2448 were pre-mixed with anti-mouse IgG (mAbZAP) oranti-human IgG (HZAP) saporin conjugates, respectively. Cancer cellswere incubated with mixtures, primary mAb alone (2448 or ch2448),saporin conjugate alone (mAbZAP or HZAP) or buffer as a control. Post 72h incubation, live cells were measured as a percentage of the controlcells treated with buffer alone. Cytotoxicity was observed by asignificant difference in live cells treated with mAbs in-complex withsecondary conjugates compared to those treated with primary mAb orsecondary conjugate alone. Results are represented as mean±standarddeviation of three independent experiments with triplicate wells (*,P<0.05; **, P<0.01; and ***, P<0.001, unpaired t-test). T47D cells weretested once in quadruplicate wells.

FIG. 19: Dose-dependent cytotoxicity of ch2448-saporin (ch2448ATS).Cells were incubated with either ch2448ATS or control (Human Igsaporin-conjugate or chTNA2ATS). (A) Cell proliferation of IGROV1 andIOSE523 cultures were monitored in real time with the xCELLigence System(Roche). Sustained inhibition of cell growth was observed on targetIGROV1 cells but not on control IOSE2523 cells. (B) Cytotoxicity ofch2448ATS was observed in a dose-dependent manner. Post 72 h, the halfmaximal inhibitory concentration was measured in IGROV1 and SKOV3cultures.

FIG. 20: ADCC activity of ch2448. ch2448 exhibited ADCC activity against(A) ovarian and (B) breast cancer cells. ADCC activity was measured asfold induction of the NFAT ADCC pathway using an ADCC reporter biossay(Promega).

FIG. 21: In vivo activity of ch2448 in a nude mouse xenograft model. (A)ch2448 suppressed IGROV1 tumor cell growth. Treatment of ch2448 (1 mg)or buffer vehicle control (↓) was administered via intraperitonealinjection. (B) No drastic weight loss was observed throughout treatmentregime. All values and bars are represented as standard error of themean (S.E.M.) Two-sided unpaired Student's t-test. *, **, and ***indicate p<0.05,0.01 and 0.005.

FIG. 22: In vitro and in vivo effects of ch2448-F(ab)2. (A) IdeSdigestion was used to generate F(ab′)2 fragments of ch2448. Digestionproducts were separated by SDS-PAGE and visualized with Coomassie Bluestaining(B) After digestion, the loss of ADCC activity was observedcompared to ch2448, in vitro. (C) In vivo, F(ab′2) did not demonstrateany suppression of tumor growth.

FIG. 23: Enhanced ADCC activity of an afucosylated ch2448. (A) Similarbinding profiles of afucosylated ch2448 and ch2448 was demonstrated byflow cytometry analysis. (B) Afucosylated ch2448 exhibited enhanced (>10fold) ADCC activity compared to the wild-type. ADCC activity wasmeasured as fold induction of the NFAT ADCC pathway using an ADCCreporter bioassay (Promega). Compared to the original ch2448, to elicitthe same ADCC effect, the concentration of afucosylated ch2448 requiredis more than 100× less.

FIG. 24: mAb 2448 in hESC Therapy. (A) mAb 2448 was raised against hESCand specifically binds to hESC but not differentiated embryoid bodies(EB). (B) As a naked antibody, mAb 2448 is able to prevent the formationof teratoma in vivo.

FIG. 25 Internalization of ch2448 in hESC. ch2448 internalizes into hESCas observed in the pH-Rodo assay. (A) ch2448 internalizes intoundifferentiated hESC as observed in the pH-Rodo assay but not intodifferentiated cells ch2448 can potentially be used as an ADC tospecifically eliminate undifferentiated hESC.

(B) As an ADC, ch2448 kills undifferentiated hESC in vitro but does notkill differentiated EBs. ch2448 can potentially be used as an ADC tospecifically eliminate undifferentiated hESC in regenerative therapy.(C) F(ab′)2 ch2448 can also be used as an ADC to specifically eliminateundifferentiated hESC in vitro.

FIG. 26. The ability of ch2448 to prevent or delay teratoma formation.(A) Single-cell suspension of human embryonic stem cells (5×10⁶ cellsper animal) were pre-incubated with either buffer (upper figure) or theADC at 4° C. for 20 minutes (lower figure) and then injected into theright hind leg muscle of SCID mice (n=3). Teratoma formation was thenevaluated with a grading method developed previously. (B) Single-cellsuspension of human embryonic stem cells (5×10⁶ cells per animal) wereinjected into the right hind leg muscle of SCID mice (n=3). Buffer(upper figure) and ADC (lower figure) were administeredintra-peritoneal. Teratoma formation was then evaluated with a gradingmethod developed previously.

FIG. 27. Structure of CAR(2448). CAR(2448) was constructed as a 2ndgeneration CAR using the CD28 co-stimulatory domain, and an IgG4 Fcregion linker domain. A T2A element and eGFP were inserted downstream inthe same open reading frame as the CAR construct.

FIG. 28. Overview of cytokine secretion by CAR-T cells. Where indicated,T cells were co-incubated with target IGROV-1 cells at a 10:1 ratio.Cells were incubated for 6 hours at 37° C., 5% CO2 before capture withanti-cytokine beads. Results indicate a significant increase in T cellcytokines by CAR(2448) T cells upon exposure to IGROV-1 cells thatexpress the target Annexin A2. In particular, granulocyte-macrophagecolony-stimulating factor, interferon-γ, interleukin-2, and tumornecrosis factor-α, are the cytokines with the highest levels ofsecretion upon activation with target.

FIG. 29. Detailed Cytokine secretion profile by CAR-T cells. Whereindicated, T cells were co-incubated with target IGROV-1 cells at a 10:1ratio. Cells were incubated for 6 hours at 37° C., 5% CO2 before capturewith anti-cytokine beads. The results indicated that multiple cytokines(GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17A, and TNF-α) showeda significant increase in cytokine production compared to a non-targetspecific CAR control. Condition 1: CAR(anti-CD19):IGROV-1; Condition 2:CAR(2448):IGROV-1; Condition 3: CAR(anti-CD19); Condition 4: CAR(2448);Condition 5: IGROV-1 (n=2 for all samples; n.s.—P>0.05; *—<0.05;**—P<0.01; ***—P<0.001)

FIG. 30. Cytotoxicity of CAR(2448). CAR(2448) mediates T cellcytotoxicity against target cells compared to non-specific CAR control.Effector IGROV-1 cells were co-incubated with CAR-T cells at varying E:Tratios, in a 4 hour assay, before detection of LDH by lysed cells. Theresults indicate that T cells nucleofected to express the CAR(2448)construct are capable of mediating cytotoxicity against target IGROV-1cells in a dose-dependent manner, compared to a non-target specific CARconstruct. (n=3 for all samples; *—P<0.05; **—P<0.01).

FIG. 31. Growth inhibition of CAR(2448). CAR(2448) T cells mediatesgrowth inhibition of target cells. Target IGROV-1 cells wereco-incubated with CAR-T cells at varying E:T ratios, and cell growth ofadherent IGROV-1 cells was monitored via electrical impedance on thexCELLigence system. The results suggest that CAR(2448) T cells arecapable of inhibiting the growth of target IGROV-1 cell in adose-dependent, time-dependent manner, with the greatest rate of growthinhibition mediated by the highest CAR(2448) T cell dose. All doselevels of CAR(2448) T cells show inhibition of target cell growthcompared to a non-target specific CAR control.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In a first aspect, there is provided an antigen-binding protein, or anantigen-binding fragment thereof. The antigen-binding protein, or anantigen-binding fragment thereof comprises (i) a heavy chain variabledomain comprising a VHCDR1 having the amino acid sequence GYSITSGYSWH(SEQ ID NO: 9); a VHCDR2 having the amino acid sequence YIHYSGSTKYNPSLKS(SEQ ID NO: 10) and a VHCDR3 having the amino acid sequence GSNYGFDY(SEQ ID NO: 11); and (ii) a light chain variable domain comprising aVLCDR1 having the amino acid sequence KSSQSLLYSNDQKNYLA (SEQ ID NO: 12),a VLCDR2 having the amino acid sequence WASIRES (SEQ ID NO: 13), and aVLCDR3 having the amino acid sequence QQYYIYPLT (SEQ ID NO: 14).

The antigen-binding protein, or antigen-binding fragment thereof, maycomprise heavy and light chain CDR regions that are about 80%, about85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99%identical to the heavy and light chain CDR regions of (i) and (ii).

In one embodiment, the heavy chain variable region comprises the aminoacid sequence set forth in SEQ ID NO:1. Alternatively, the heavy chainvariable region may comprise an amino acid sequence having about 80%,about 85%, about 90%, about 95%, about 96%, about 97%, about 98% orabout 99% identity to the amino acid sequence set forth in SEQ ID NO:1.

In one embodiment, the light chain variable region may comprise theamino acid sequence set forth in SEQ ID NO:2. Alternatively, the lightchain variable region may comprise an amino acid sequence having about80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% orabout 99% identity to the amino acid sequence set forth in SEQ ID NO:2.

In another aspect, there is provided an antigen-binding protein, or anantigen-binding fragment thereof, comprising (i) a heavy chain variabledomain comprising a VHCDR1 having the nucleic acid sequenceGCTACTCCATCACCAGTGGTTATAGCTGGCAC (SEQ ID NO: 15); a VHCDR2 having thenucleic acid sequence ACATACACTACAGTGGTAGCACTAAGTACAACCCATCTCTCAAAAGTC(SEQ ID NO: 16), and a VHCDR3 having the nucleic acid sequenceGGAGTAACTACGGATTTGACTACT (SEQ ID NO: 17); and (ii) a light chainvariable domain comprising a VLCDR1 having the nucleic acid sequenceAGTCCAGTCAGAGCCTTTTATATAGTAACGATCAAAAGAACTACTTGGCCT (SEQ ID NO: 18), aVLCDR2 having the nucleic acid sequence GGGCATCTATTAGGGAATCTG (SEQ IDNO: 19), and a VLCDR3 having the nucleic acid sequenceAGCAATATTATATCTATCCTCTCACGT (SEQ ID NO: 20).

In one embodiment, the antigen-binding protein, or antigen-bindingfragment thereof, may comprise heavy and light chain CDR regions thatare about 60%, 65%, 70%, 75%, 80%, about 85%, about 90%, about 95%,about 96%, about 97%, about 98% or about 99% identical to the heavy andlight chain CDR regions of (i) and (ii).

In one embodiment, the heavy chain variable region may comprise thenucleic acid sequence set forth in SEQ ID NO:3. Alternatively, theantigen-binding protein, or antigen-binding fragment thereof, maycomprise a heavy chain variable region which comprises a nucleic acidsequence having about 60%, 65%, 70%, 75%, 80%, about 85%, about 90%,about 95%, about 96%, about 97%, about 98% or about 99% identity to thenucleic acid sequence set forth in SEQ ID NO:3.

In one embodiment, the light chain variable region may comprise thenucleic acid sequence set forth in SEQ ID NO:4. Alternatively, theantigen-binding protein, or antigen-binding fragment thereof, maycomprise a light chain variable region which comprises a nucleic acidsequence having about 60%, 65%, 70%, 75%, 80%, about 85%, about 90%,about 95%, about 96%, about 97%, about 98% or about 99% identity to theamino acid sequence set forth in SEQ ID NO:4.

In one embodiment the antigen-binding protein, or antigen-bindingfragment thereof, may be selected from the group consisting ofmonoclonal, recombinant, polyclonal, chimeric, humanised, bispecific andheteroconjugate antibodies; a chimeric antigen receptor (CAR), a singlevariable domain, a domain antibody, antigen binding fragments,immunologically effective fragments, single chain Fv, a single chainantibody, a univalent antibody lacking a hinge region, a minibody,diabodies, and Tandabs™.

In one embodiment, the binding protein may be a monoclonal antibody. Themonoclonal antibody may be 2448. In one embodiment, the monoclonalantibody may be humanised. Alternatively, the monoclonal antibody may bechimeric.

The monoclonal antibody may be defucosylated. The degree of fucosylationis may be less than 10%, less than 5%, or less than 1.5% relative to thewild-type antibody.

In another aspect there is provided an antigen-binding protein, or anantigen-binding fragment thereof, comprising (i) a heavy chain variabledomain comprising a VHCDR1 having the amino acid sequence VYSITSGYSWH(SEQ ID NO: 21); a VHCDR2 having the amino acid sequenceYIHYSGSTKYNPSLKS (SEQ ID NO: 10), and a VHCDR3 having the amino acidsequence GTDNAVDY (SEQ ID NO: 22); and (ii) a light chain variabledomain comprising a VLCDR1 having the amino acid sequenceKSSQSLLYSSNQKNYLA (SEQ ID NO: 23), a VLCDR2 having the amino acidsequence WASSRES (SEQ ID NO: 24), and a VLCDR3 having the amino acidsequence QQYYIYPLT (SEQ ID NO: 14).

In one embodiment, the antigen-binding protein, or antigen-bindingfragment thereof, may comprise heavy and light chain CDR regions thatare about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98% or about 99% identical to the heavy and light chain CDRregions of (i) and (ii).

In one embodiment, the heavy chain variable region may comprise theamino acid sequence set forth in SEQ ID NO:5. Alternatively, the heavychain variable region may comprise an amino acid sequence having about80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% orabout 99% identity to the amino acid sequence set forth in SEQ ID NO:5.

In one embodiment, the light chain variable region may comprise theamino acid sequence set forth in SEQ ID NO:6. Alternatively, theantigen-binding protein, or antigen-binding fragment thereof, maycomprise a light chain variable region which comprises an amino acidsequence having about 80%, about 85%, about 90%, about 95%, about 96%,about 97%, about 98% or about 99% identity to the amino acid sequenceset forth in SEQ ID NO:6.

In another aspect, there is provided an antigen-binding protein, or anantigen-binding fragment thereof, comprising (i) a heavy chain variabledomain comprising a VHCDR1 having the nucleic acid sequenceTCTACTCCATCACCAGTGGTTATAGCTGGCACT (SEQ ID NO: 25); a VHCDR2 having thenucleic acid sequence ACATACACTACAGTGGTAGTACTAAGTACAACCCATCTCTCAAAAGTC(SEQ ID NO: 26), and a VHCDR3 having the nucleic acid sequenceGGACCGACAATGCTGTGGACTACT (SEQ ID NO: 27); and (ii) a light chainvariable domain comprising a VLCDR1 having the nucleic acid sequenceAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCT (SEQ ID NO: 28), aVLCDR2 having the nucleic acid sequence GGGCATCCAGTAGGGAATCTG (SEQ IDNO: 29), and a VLCDR3 having the nucleic acid sequenceAGCAATATTATATCTATCCTCTCACGT (SEQ ID NO: 20).

The antigen-binding protein, or antigen-binding fragment thereof, maycomprise heavy and light chain CDR regions that are about 60%, 65%, 70%,75%, 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about98% or about 99% identical to the heavy and light chain CDR regions of(i) and (ii).

The heavy chain variable region may comprise the nucleic acid sequenceset forth in SEQ ID NO:7. The heavy chain variable region may comprise anucleic acid sequence having about 60%, 65%, 70%, 75%, 80%, about 85%,about 90%, about 95%, about 96%, about 97%, about 98% or about 99%identity to the nucleic acid sequence set forth in SEQ ID NO:7.

The light chain variable region may comprise the nucleic acid sequenceset forth in SEQ ID NO:8. The antigen-binding protein, orantigen-binding fragment thereof, may comprise a light chain variableregion which comprises a nucleic acid sequence having about 60%, 65%,70%, 75%, 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98% or about 99% identity to the amino acid sequence set forth inSEQ ID NO:8.

In one embodiment, the antigen binding protein may be selected from thegroup consisting of monoclonal, recombinant, polyclonal, chimeric,humanised, bispecific and heteroconjugate antibodies; a chimeric antigenreceptor (CAR), a single variable domain, a domain antibody, antigenbinding fragments, immunologically effective fragments, single chain Fv,a single chain antibody, a univalent antibody lacking a hinge region, aminibody, diabodies, and Tandabs™.

In one embodiment, the binding protein may be a monoclonal antibody. Themonoclonal antibody may be C51. The monoclonal antibody may behumanised. Alternatively, the monoclonal antibody may be chimeric.

In one embodiment, the antigen-binding protein, or antigen-bindingfragment thereof, as described herein, may bind to ANXA2. In oneembodiment, the antigen-binding protein, or antigen-binding fragmentthereof, as described herein, may bind to a glycan on ANXA2. Theantigen-binding protein, or an antigen-binding fragment thereof, asdescribed herein, may bind to an N-linked glycan on ANXA2. The N-linkedglycan may be located at amino acid residue 62 of ANXA2.

In another embodiment, the antigen-binding protein, or antigen-bindingfragment thereof, as described herein, may comprise a radioisotope or acytotoxin conjugated thereto. The antibody may be conjugated with acytotoxin selected from the group consisting of monomethyl auristatin E(MMAE-1), mertansine (DM-1) and saporin.

In one embodiment, the antigen-binding protein, or an antigen-bindingfragment, as described herein, may be internalized into a cell uponbinding to ANXA2.

In one embodiment, the antigen-binding protein, or an antigen-bindingfragment thereof, as described herein, may have a cytotoxic activityselected from one or more of the group consisting of complementdependent cytotoxic (CDC) activity, antibody dependent cellularcytotoxic (ADCC) activity and oncolytic activity.

In another aspect, there is provided a composition comprising aphysiologically acceptable carrier and a therapeutically effectiveamount of the antigen-binding protein, or an antigen-binding fragmentthereof, as described herein.

In one embodiment, the composition may comprise a further activepharmaceutical ingredient selected from the group consisting ofbevacizumab, carboplatin, paclitaxel or gefitinib.

The percentage of the antigen-binding protein, or an antigen-bindingfragment thereof, as described herein, in pharmaceutical compositionsand preparations may, of course, be varied and, for example, mayconveniently range from about 2% to about 90%, about 5% to about 80%,about 10% to about 75%, about 15% to about 65%; about 20% to about 60%,about 25% to about 50%, about 30% to about 45%, or about 35% to about45%, of the weight of the dosage unit. The amount of compound intherapeutically useful compositions is such that a suitable dosage willbe obtained.

The language “physiologically acceptable carrier” is intended to includesolvents, dispersion media, coatings, anti-bacterial and anti-fungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the compound, use thereof in the therapeuticcompositions and methods of treatment and prophylaxis is contemplated.Supplementary active compounds may also be incorporated into thecompositions according to the present invention. It is especiallyadvantageous to formulate parenteral compositions in dosage unit formfor ease of administration and uniformity of dosage. “Dosage unit form”as used herein refers to physically discrete units suited as unitarydosages for the individual to be treated; each unit containing apredetermined quantity of compound(s) is calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The compound(s) may be formulated for convenientand effective administration in effective amounts with a suitablepharmaceutically acceptable carrier in an acceptable dosage unit. In thecase of compositions containing supplementary active ingredients, thedosages are determined by reference to the usual dose and manner ofadministration of the said ingredients.

The composition may be conveniently administered by injection, forexample, subcutaneous, intravenous, and the like. The composition mayalso be administered parenterally or intraperitoneally. In oneembodiment, the compound may be administered by injection. In the caseof injectable solutions, the carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyetheylene glycol, and thelike), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by including various anti-bacterialand/or anti-fungal agents. Suitable agents are well known to thoseskilled in the art and include, for example, parabens, chlorobutanol,phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like. In manycases, it may be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theanalogue in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilisation. Generally, dispersions are prepared byincorporating the analogue into a sterile vehicle which contains a basicdispersion medium and the required other ingredients from thoseenumerated above.

Under ordinary conditions of storage and use, pharmaceuticalpreparations may contain a preservative to prevent the growth ofmicroorganisms. Preferably, the pharmaceutical composition may furtherinclude a suitable buffer to minimise acid hydrolysis. Suitable bufferagent agents are well known to those skilled in the art and include, butare not limited to, phosphates, citrates, carbonates and mixturesthereof.

Single or multiple administrations of the pharmaceutical compositionsaccording to the invention may be carried out. One skilled in the artwould be able, by routine experimentation, to determine effective,non-toxic dosage levels of the compound and/or composition of theinvention and an administration pattern which would be suitable fortreating the diseases and/or infections to which the compounds andcompositions are applicable.

Further, it will be apparent to one of ordinary skill in the art thatthe optimal course of treatment, such as the number of doses of thecompound or composition of the invention given per day for a definednumber of days, can be ascertained using convention course of treatmentdetermination tests.

Generally, an effective dosage per 24 hours may be in the range of about0.0001 mg to about 1000 mg per kg body weight; suitably, about 0.001 mgto about 750 mg per kg body weight; about 0.01 mg to about 500 mg per kgbody weight; about 0.1 mg to about 500 mg per kg body weight; about 0.1mg to about 250 mg per kg body weight; or about 1.0 mg to about 250 mgper kg body weight. More suitably, an effective dosage per 24 hours maybe in the range of about 1.0 mg to about 200 mg per kg body weight;about 1.0 mg to about 100 mg per kg body weight; about 1.0 mg to about50 mg per kg body weight; about 1.0 mg to about 25 mg per kg bodyweight; about 5.0 mg to about 50 mg per kg body weight; about 5.0 mg toabout 20 mg per kg body weight; or about 5.0 mg to about 15 mg per kgbody weight.

Alternatively, an effective dosage may be up to about 500 mg/m². Forexample, generally, an effective dosage is expected to be in the rangeof about 25 to about 500 mg/m², about 25 to about 350 mg/m², about 25 toabout 300 mg/m², about 25 to about 250 mg/m², about 50 to about 250mg/m², and about 75 to about 150 mg/m².

In another aspect, there is provided use of an antigen-binding protein,or an antigen-binding fragment thereof, as described herein, in themanufacture of a medicament for treating or preventing cancer.

In one embodiment, the cancer may be selected from the group consistingof breast cancer, liver cancer, kidney cancer, colorectal cancer,ovarian cancer and teratoma.

In some embodiments the medicament may be administered with a furtheractive pharmaceutical ingredient. Alternatively, the medicament may beadministered with chemotherapy. The further pharmaceutical agent orchemotherapy may be administered separately, simultaneously orsequentially.

In another aspect, there is provided a method for detecting cancer in asubject, the method comprising: contacting a sample obtained from thesubject with an antigen-binding protein, or an antigen-binding fragmentthereof as described herein in vitro; detecting the binding of theantigen-binding protein, or an antigen-binding fragment thereof in thesample; correlating the binding with a level of binding in a controlsample to determine the level of binding in the sample, wherein anincrease in the level of binding in the sample relative to the controlsample is indicative of cancer.

In another aspect, there is provided a method for identifying a subjectsusceptible to cancer the method comprising: contacting a sampleobtained from the subject with an antigen-binding protein, or anantigen-binding fragment thereof as described herein in vitro; detectingthe binding of the antigen-binding protein, or an antigen-bindingfragment thereof in the sample; correlating the binding with a level ofbinding in a control sample to determine the level of binding in thesample, wherein an increase in the level of binding in the samplerelative to the control sample indicates that the subject is susceptibleto cancer.

In one embodiment, the control sample may be from the same subject.Alternatively, the control sample may be from a different subject.

In one embodiment, the antigen-binding protein, or antigen-bindingfragment thereof as described herein may comprise a detectable label.The detectable label may be selected from the group consisting of afluorescent label, a chemiluminescent label, an enzymatic label and aradionuclide label. In one embodiment, the detectable label is selectedfrom the group consisting of biotin, alkaline phosphatase, horseradishperoxidase, FITC, PE and Cy Dyes. The detectable label may be detectedin an assay selected from flow cytometry, tissue section,immunofluorescence, immunocytochemistry or immunohistochemistry.

In one aspect, there is provided a kit when used in the method asdescribed herein, comprising an antigen-binding protein, orantigen-binding fragment thereof as described herein, together withinstructions for use.

EXAMPLES

Non-limiting examples of the invention, including the best mode, and acomparative example will be further described in greater detail byreference to specific Examples, which should not be construed as in anyway limiting the scope of the invention.

Materials and Methods

Antibody Generation and Purification

Monoclonals 2448 and C51 were generated by whole-cell immunization ofhuman embryonic stem cells (HES-3) using mouse hybridoma technology asreported in Choo et al. (Choo A B, Tan H L, Ang S N, Fong W J, Chin A,Lo J, et al. Selection against undifferentiated human embryonic stemcells by a cytotoxic antibody recognizing podocalyxin-like protein-1.Stem Cells Dayt Ohio. 2008; 26:1454-63). Hybridomas were maintained inClonaCell™-HY Medium E (Stem Cells Technologies) at 37° C. in ahumidified incubator with 5% CO₂. Chimeric (including afucosylated)antibodies were expressed in DG44-CHO cells and maintained in BTI'sproprietary serum-free media. Chimerisation was done by the Animal CellTechnology group at the Bioprocessing Technology Institute (Ho S C L,Bardor M, Feng H, Mariati null, Tong Y W, Song Z, et al. IRES-mediatedTricistronic vectors for enhancing generation of high monoclonalantibody expressing CHO cell lines. J Biotechnol. 2012; 157:130-9).

Purification was done using the AKTA Explorer 100 (GE Healthcare)system. Cultured supernatants were subjected to Protein A chromatography(Tosoh; Toyopearl AF-rProtein A-650F) and ion exchange chromatography(Biorad; UNOsphere™ Q). Purified products were evaluated on aSuperdex200 PC 3.2/30 column (GE Healthcare) using a high performanceliquid chromatography system (Shimadzu). Antibodies were additionallyanalyzed by SDS-PAGE, and protein concentrations were determined byabsorbance at A₂₈₀ using Nanodrop 1000 (Thermo Fisher Scientific). Forin vivo studies, endotoxin was measured to ensure levels were below 0.1EU/mL using the Endosafe® Endotoxin Testing System (Charles River).

Flow Cytometry and Analysis

Cells were harvested as single-cell suspensions using trypsin (ThermoFisher Scientific). Each sample of 1-2×10⁵ cells was thoroughly washedin 1% bovine serum albumin (BSA; Sigma Aldrich) in phosphate bufferedsaline (PBS) buffer (Thermo Fisher Scientific) Samples were incubatedwith primary antibody at 4° C. for 45 min, washed and then incubatedwith the appropriate fluorophore-conjugated secondary antibody(fluorescein isothiocyanate (FITC)-labeled goat anti-human kappa lightchain mAbs (Sigma Aldrich) or FITC-labeled goat anti-mouse Ig polyclonal(Dako), for 15 min at 4° C. After incubation, cells were washed andsample data acquisition was done by on a BD FACSCalibur™ (BDBiosciences) or Guava® easyCyte (Millipore). Data analysis was doneusing FlowJo™ software v7.6.3 (Tree Star). Percentage of binding wasdetermined using M-gating set at the 97th-98th-percentile based on thenegative control.

Immunocytochemistry

Cell cultures were fixed with 4% paraformaldehyde for 20 min. Forpermeabilized cells, 0.1% Triton X-100/PBS (Bio-Rad) was added for 3×5min, and finally blocked with either 10% goat serum/PBS (DAKO) for 1 hat room temperature. Cells were incubated at 4° C. with 2448 conjugatedwith DyLight™ 488 NHS Ester (Thermo Fisher Scientific). DNA wascounterstained with DAPI (1:1000, Thermo Fisher Scientific). Images weretaken using a Zeiss Axiovert 200 inverted microscope.

Gel Electrophoresis and Western Blot Analysis

Cells were harvested by manual scraping and as required, enriched formembrane proteins via the Membrane Protein Extraction Kit (BioVision) asper the manufacturer's instructions. Briefly, cells were re-suspended inhomogenization buffer and centrifuged at 700 g for 10 min at 4° C.Supernatant was then aspirated and centrifuged at 10,000 g for 30 min at4° C. Cell pellet of total membrane proteins was collected and lysed.Buffer for lysis contained 2% Triton X-100 (Bio-Rad) in PBS which wassupplemented with protease inhibitors (Calbiochem) as needed. Totalprotein concentration was determined using the DC™ Protein Assay(Bio-Rad).

For gel electrophoresis, samples were prepared with loading dye at afinal concentration of 50 mM Tris-HCl, 2% SDS, 10% glycerol, 0.02%bromophenol blue and for reducing conditions, 2-5% beta-mercaptoethanol.Samples along with SeeBlue Plus2® or Page2™ protein standards (ThermoFisher Scientific) were subjugated to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) using a 4-12% Bis-Trisgradient gel (Thermo Fisher Scientific) and 1× MOPS buffer (ThermoFisher Scientific). If needed, Gels were stained using CoomasieBrilliant Blue or SilverQuest™ Silver Staining Kit)

For Western blot, products from gel run were transferred topolyvinylidene difluoride (PVDF) membranes (Bio-Rad) and blocked with 5%non-fat milk or Odyssey® Blocking Buffer (LI-COR) for 1 h. Incubationwith primary mAb (mouse anti-annexin A2 mAb (BD Biosciences); mouseanti-annexin A2 mAb (Invitrogen); rabbit anti-annexin A2 pAb (SantaCruz); mouse anti-E-Cadherin mAb (BD Biosciences); mouse monoclonalanti-Vimentin (Dako); mouse anti-human Fc-specific antibody (Sigma)) orbiotinylated lectin (Aleuria Aurantia Lectin (Vector Labs)) was doneovernight at 4° C. The blot was incubated for 1-2 h with an appropriatedetection antibody (horseradish peroxidase (HRP)-conjugated goatanti-mouse Ig pAb, Dako) or streptavidin conjugated to horseradishperoxidase (Dako), and visualized upon addition of chemiluminescencesubstrate (GE Healthcare).

Images were captured either on Medical X-ray Processor 2000 (Kodak) orthe ChemiDoc™ Imaging System (Bio-Rad). Densitometry was done usingImage Lab™ 5.2 software (BioRad) and normalized to beta-actin expressionlevels (Cell Signaling Technology).

Transient Knockdown of Annexin A2 (ANXA2)

IGROV1 cells were seeded on 6-well plates at 3×10⁵ cells or T175 flasksat 1×10⁶ cells and grown to 30 to 50% confluency. Transfection of avalidated set of human annexin A2-specific siRNA (Thermo FisherScientific) and scramble siRNA control (Thermo Fisher Scientific)respectively both at concentration of 30 pM using Lipofectamine RNAiMAX(Thermo Fisher Scientific) in serum-free media. Post 5 h incubation at37° C., transfection media was removed and replaced with fresh mediacontaining 10% serum. Cells were harvested within 72 h for western blotanalysis. Densitometry was done using IMageJ software (NationalInstitutes of Health) and normalized using beta-actin (Cell SignalingTechnology) expression levels.

Periodate Treatment

Lysate from IGROV1 cells was separated by SDS-PAGE and transferred toPVDF membranes (Bio-Rad). Subsequently, blots were washed twice withsodium acetate buffer (100 mM at pH 4.5; Merck Millipore), and incubatedwith sodium meta-periodate (100 mM, Sigma-Aldrich) for 30 min in thedark. Blots were subsequently washed four times with sodium acetatebuffer, a PBS wash and quenched with 0.5 M of sodium borohydride (Sigma)for 30 min. Prior to incubation with primary antibodies, blots wereblocked with Odyssey® Blocking Buffer (LI-COR) for 30 min. Control blotswere similarly incubated with buffers but without the addition of sodiummeta-periodate. Samples were analyzed using primary antibodies 2448, C51and anti-beta-actin as a negative control.

Release of Glycans by PNGase-F and Beta-Elimination

For PNGase-F treatment, membrane protein enriched lysate (10 μg) wasfirst denatured using 1 μl of 10× glycoprotein denaturing buffer (5% SDSwithout DTT) and water to make up a 10 μl reaction volume. Glycoproteinswere heated to 100° C. for 5 min. A total reaction volume of 20 μl wasprepared with the denatured glycoproteins by adding 2 μl of 10×G7reaction buffer, 2 μl of 10% NP40, and 1 μl of PNGase F, and 5 μl ofwater. Samples were incubated in the reaction buffer for 1 h at 37° C.After incubation, samples were subjected to SDS-PAGE and western blotanalysis. For alkaline beta-elimination treatment, western blots wereincubated in 0.1 M sodium hydroxide solution at 60° C. overnight. Thefollowing day blots were subjected to western blot analysis.

Inhibition of Glycosylation by Tunicamycin

Inhibition of glycosylation was done in cell culture using tunicamycin,a nucleoside antibiotic that targets GlcNAc transferases. At highconcentrations, tunicamycin can induce apoptosis in cancer cells howeverat lower concentrations, it can be used to inhibit synthesis of N-glycanin (eukaryotic) cells (127-129). IGROV1 cells at 60-70% confluency weretreated with tunicamycin (Sigma-Aldrich) at a final concentration of0.25 μg/mL. After 24 h incubation, cells harvested and analyzed by flowcytometry and western blot analysis.

Competitive Inhibition Assay

IGROV1 cells were incubated at saturating concentrations of a singleantibody (2448 or ch2448) added alone, both antibodies addedsimultaneously and one mAb added after the other. Incubations were donefor at least 15 min each at 4° C. and washed in 1% BSA/PBS. Secondaryincubation was done using fluorophore conjugated secondary antibodies(Alexa fluor 647-conjugated anti-human pAb (Thermo Fisher Scientific)and Alexa Fluor 488-conjugated anti-mouse pAb (Thermo FisherScientific)). Binding was analyzed on a BD FACSCalibur™ flow cytometer(BD Biosciences) or Guava® easyCyte (Millipore).

Real-Time Monitoring of 2448-Saporin Cytotoxicity on Cell Growth

Cell growth was continuously cells was determined when cells treated atthe lowest concentration were reaching a monitored over time by cellimpedance measurements using the xCelligence® real-time cell analyzer(Roche) (130). Briefly, cell culture media was first loaded onto the96-well E-plate to measure background impedance. IGROV1, IOSE523 andSfilm.

V3 cells were plated at 1,000 cells per well and allowed to growovernight in normal cell culture conditions. Cells were treated withantibody (ch2448) or antibody conjugates (ch2448-saporin or humanIgG-saporin) at the beginning of the log phase of cell growth. Controlwells were treated with buffer alone. All experiments were done in atleast 5 wells per treatment condition. The outermost wells of the platewere not used. For dose response curves, IGROV1 were treated withdilutions of ch2448-saporin. The cells were monitored for growth untilcells reached a death phase. Cell indices of wells were normalized afterantibody treatment. The IC₅₀ value on IGROV1 stationary phase of cellgrowth. The IC₅₀ value was calculated using the accompanying real-timecell analysis software (Roche).

Antibody Drug Conjugate (ADC) Assay with Secondary Saporin Conjugates

Cells were seeded in 96-well culture plates (Corning) at 1000 or 2000cells per well as determined by growth curve. Primary antibody (2448,C51, ch2448 or chC51) at 10 μg/ml were complexed with appropriatesecondary saporin conjugates: mAb-ZAP, Anti-M-ZAP, HUM-ZAP (AdvancedTargeting Systems), at a 1:3 molar ratio for 15 min at room temperature.The pre-mixed complexes, the primary mAb, the secondary conjugate andbuffer control were added to wells 24 h post-seeding. At 72 hpost-treatment, viable (metabolically active) cells were measured basedon the presence of ATP, using the CellTiter-Glo® Luminescent CellViability Assay kit (Promega). Data are expressed as the viability oftreated cells as the percentage of the control cells treated with bufferalone.

Antibody Drug Conjugate (ADC) Assay with ch2448-Saporin Conjugate

Direct conjugation of saporin to antibodies was outsourced to AdvancedTargeting Systems (ATS). Drug to antibody molar ratios ofch2448-saporin, chTNA2-saporin and human Ig-saporin were 2.5, 2.9 and3.1, respectively. Evaluation of the binding specificity was carried outby flow cytometry analysis as previously described.

Cytotoxicity of ch2448-saporin was evaluated on IGROV1, SKOV3 andIOSE523 cell lines. Cells were seeded in 96-well plates (Corning) at1000 cells per well in 90 μl of media. The following day, ch2448-saporinand chTNA2-saporin were serially diluted and 10 μl of each dilution wasadded to wells. As a control, free saporin and ch2448 were added in aseparate set of plates. Post 72 h incubation, the cell viability wasmeasured using the CellTiter-Glo® Luminescent Cell Viability Assay kit(Promega) according to the manufacturer's instructions. Data areexpressed as the % control, measuring the viability of treated cellswith that of untreated cells. Dose response curves and IC₅₀ values werecalculated using GraphPad Prism 6 (GraphPad).

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Assay

ADCC activity was measured using the ADCC Reporter Bioassay reporterbioassay (Promega) according to the manufacturer's protocol. Briefly,cells were seeded at 5,000 cells per well in a 96-well clear bottomblack tissue culture plates (Corning) in low 4% IgG-serum (Promega)media. Serial dilutions of primary antibody were incubated in triplicatewells for approximately 15 min at 37° C., 5% CO2. Following incubation,engineered effector cells were added to the wells at approximately150,000 cells per well. After 5 to 16 h (as indicated in results),Bio-Glo™ Luciferase Assay Substrate (Promega) was added to the wells andluminescence was measured using the Infinite® 200 microplate reader(Tecan). Estimated EC₅₀ values were calculated by on-linear regressionon log-transformed data using GraphPad Prism 6 (GraphPad).

Tumor Xenograft Mouse Model

Four- to six-week-old female BALB/c nude mice (Invivos) were used inthis study. On day 0, mice were inoculated with a total of 5×10⁶ IGROV1cells in 100 μl of cell media and high concentration matrigel (BDbiosciences) at a 1:1 dilution volume. At 24 h post-inoculation,antibodies were injected intraperitoneally at 100 μl volumes of 1 mg perdose in 50 mM HEPES, 150 mM NaCl buffer. Subsequent administration wasdone weekly as indicated in results for 5 weeks. Size of the primarytumor was measured weekly by digital calipers. Tumor volumes (TV) werecalculated based on the formula: TV=((L×L×W)/2), where W (width) and L(length) are the short and long diameter, respectively. A student'st-test was used to assess the statistical significance treated anduntreated animals. For collection of tumors, animals were euthanized andimmediately harvested for tumors. Samples were washed with PBS andeither snap-frozen in liquid nitrogen or placed in paraformaldehyde(Merk) for 24 h which was subsequently replaced with 100% ethanol(Merk). Mice were euthanized when tumor size was >2000 mm³ or whenpersistent side effects (e.g. swollen lymph nodes or drastic body weightloss) were observed over a period of more than two weeks. Euthanizationwas done by CO₂ inhalation followed by cervical dislocation. Animalswere handled according to Biopolis IACUC Protocol No.:151001 inaccordance with the National Advisory Committee For Laboratory AnimalResearch (NACLAR) Guidelines.

Biodistribution Study

Female BALB/c nude mice were inoculated with a total of 5×10⁶ IGROV1cells in 100 μl of cell media and high concentration matrigel (BDbiosciences) at a 1:1 dilution volume. When tumors reached 300-400 mm³(on week 10), dye-conjugated ch2448 and control conjugate chTNB1 wereadministered by i.p. injection at 100 μg per 100 μl of buffer (50 mMHEPES, 150 mM NaCl) per mouse. At 72 h and 94 h post injection, micewere anesthetized with 2-3% isoflurane and imaged using the IVIS®Spectrum imaging system (Caliper Life Sciences). Data was analyzed usingthe Living Image software 3.2 (Caliper Life Sciences).

Antibodies ch2448 and IgG control (Southern Biotech) were labelled witha near infrared fluorescent (NIR) dye CF750 using the XenoLight CF750rapid antibody-labelling kit (Caliper Life Sciences) as per themanufacturer's instructions. Mice were handled according to BiopolisIACUC Protocol No.: 151001 in accordance with the National AdvisoryCommittee For Laboratory Animal Research (NACLAR) Guidelines.

CAR Design and Vectors

CAR(2448) was designed in silico, and manufactured by GenScript. The CARconstruct contained the GMCSFRa signal sequence, the anti-Annexin A2V_(L) and V_(H) domains of the 2448 antibody connected via a Whitlowlinker, the IgG₄ Fc linker region, the CD28 transmembrane andintracellular co-stimulatory domain, and the CD3ζ signalling domain. TheCAR fragment was synthesised into the pcDNA3.1(+) plasmid, using HindIIIand EcoRI restriction sites.

mRNA Production

mRNA of the CAR constructs was in vitro transcribed utilising theHiScribe™ T7 ARCA mRNA Kit (with tailing) (New England BioLabs),according to the manufacturer's instructions. Briefly, CAR sequenceplasmids were linearised utilising the XbaI restriction site, purified,and in vitro transcribed with ARCA capping. A subsequent poly(A) tailingreaction was conducted, before purification with lithium chloride. Thefinal mRNA constructs were reconstituted in nuclease-free water, andstored at −80° C. in single-use aliquots.

Nucleofection of T Cells

T cells were isolated from human PBMCs using an EasySep human T cellisolation kit (StemCell Technologies). T cells were activated with HumanT-Activator CD3/CD28 Dynabeads (Life Technologies) at a 1:1 bead:cellratio. RPMI 1640, supplemented with 10% foetal calf serum (R10), andIL-7(20 U/mL), IL-15(10 U/mL), and IL-21(0.04 U/mL) was utilised as theT cell culture medium.

Prior to nucleofection, activating Dynabeads were removed, and T cellswere resuspended in P3 Primary Cell Nucleofector™ Solution (Lonza) at5×10⁴ cells/μL. CAR mRNA (61 pg/μL) was added to the T cells, beforenucleofection using program EO-115 of the 4D Nucleofector™ device. Cellswere allowed to recover in R10 media at 37° C., 5% CO₂, with or withoutadditional cytokine supplementation, depending on downstreamapplication.

Cytotoxicity Assays

CAR-T cell cytotoxicity was assayed using the CytoTox 96®Non-Radioactive Cytotoxicity Assay (Promega), according to themanufacturer's instructions. Briefly, target IGROV-1 cells (1×10⁴cells/well) were co-incubated with effector CAR-T cells at varyingeffector:target ratios for 4 hours at 37° C., 5% CO₂. LDH release fromlysed cells was detected using the kit solutions, and readout using anInfinite® M200 (TECAN).

For the cell growth assay, target IGROV-1 cells were seeded onto anE-plate 96 (ACEA Biosciences) at 1×10⁴ cells per well. Cell growth wasmeasured using the xCELLigence RCPA MP Instrument (ACEA Biosciences).After 24 hours, freshly nucleofected CAR-T cells were added into eachwell at 1:1, 2:1, 4:1, and 8:1 effector:target ratios. Cell growth wasmonitored for an additional 72 hours. No effector and no target cellwells were used as controls.

Cytokine Release Assay

CAR-T cells were incubated with target IGROV-1 cell at a 10:1 ratio for6 hours at 37° C., 5% CO₂, without additional cytokine supplementation,before cytokine detection with the MACSPlex Cytokine 12 Kit (MiltenyiBiotec), according to the manufacturer's instructions. Briefly, cellsupernatants were centrifuged and collected, before the addition of MPxCytokine 12 Capture Beads. After a 2 hour incubation period at roomtemperature, capture beads were washed twice, and MPx Cytokine 12Detection Reagent was added to detect bound cytokines. The beads wereincubated for an additional hour at room temperature, before cytokinedetection on the MACS Quant Analyzer 10 flow cytometer (MiltenyiBiotec).

Results

Antibody (Ab) heavy and light chain sequences and isotype of monoclonalantibody (mAb) C51 is an IgM-kappa.

Heavy and light chain gene sequences of mAb C51 were cloned fromhybridoma cells by reverse transcription polymerase chain reaction(RT-PCR) and sequenced with complementarity-determining regions (CDRs)as underlined (FIGS. 1A and B). Isotyping of supernatant from hybridomaclone supernatant revealed that mAb C51 was an IgM-kappa immunoglobulin.

mAb C51 demonstrates reactivity to various cancers. mab C51 binds to anepithelial phenotype according to a classification based on theepithelial-mesenchymal transition (EMT).

High-throughput screening of mAb C51 was done on live cells by flowcytometry (FIG. 2A). Cell surface binding was observed on breast (BT474,BT20, CAMA1, HCC1937, HCC1954, HCC2218, MS578T, MCF7, MDAMB453, SKBR3,T47D), colorectal (COL0205, HT29, KM12, LS174T, SW620), kidney (786O,A498, ACHN, CAKI1, UO31, RXF393, TK10), liver (HEP3B, HEPG2, HUH7, PLC)and ovarian (CAOV3, IGROV1, OV90, OVCA432, OVCAR3, OVCAR8, OVCA433,PEA1, SKOV3) cancer cells. No binding was observed on normal cell lines(IMR90, IOSE523, HEK293, HFF, hTERT-HME1). Results showed that mAb C51can specifically bind to multiple cancers. Binding of mAb C51 to ovarianand breast cancer cells was organized according to anEpithelial-Mesenchymal Transition (EMT) classification (FIG. 2B). mAbC51 was shown to preferentially bind to cells classified with anepithelial (E) and intermediate epithelial (IE) phenotype. Resultsshowed that mAb C51 can potentially be used to monitor EMT.

Annexin A2 is the antigen target of mAb C51.

To validate ANXA2 as the antigen target of mAb C51, a forward andreverse-immunoprecipitation (IP) was carried out using a commercialanti-ANXA2 mAb. IP products were immunoblotted against mAb C51 and thecommercial antibody (FIG. 3A). mAb C51 recognized similar antigen bandsfor both IP products. A transient siRNA knockdown study of ANXA2 wasalso carried out (FIG. 3B). Partial knockdown of ANXA2 corresponded to aloss of antigen recognition by mAb C51. Taken together, resultsdemonstrated Annexin A2 as the antigen target of mAb C51.

mAb C51 targets an N-glycan epitope.

The binding of C51 was abolished when N-linked glycans were removed.This was demonstrated by both enzymatic digest of proteins with PNGase Ftreatment (FIG. 4A) and inhibition of N-linked glycosylation in cellswith Tunicamycin (FIG. 4B). These results demonstrated that C51 wasbinding to N-glycan-epitope on ANXA2.

mAb C51 induces cell death via oncosis.

Ovarian and breast cancer cells incubated with C51 had a significant andrapid increase in PI uptake, indicating a loss of membrane integrity(FIG. 5). A loss of relative viability was observed on IGROV1, MCF7,SKOV3 and T47D cells. Minimal cytotoxicity was observed in media-onlycontrols. Results indicated cell-death via oncosis.

mAb C51 as an antibody-drug conjugate (ADC) kills cancer cells.

Ovarian and breast cancer cells were incubated in culture with mAb C51and secondary antibody conjugated to saporin (MZAP). After 72 h ofincubation, relative cell viability was measured by the percentage oflive cells in the treatment group compared to the buffer-treated control(FIG. 6). Up to 50% cytotoxicity with mAb C51 and secondary mAbs wasdemonstrated on both IGROV1 and MCF7 cell lines compared to the buffercontrol, mAb C51 alone or saporin secondary Ab alone. No cytotoxicitywas observed for non-binding control cell lines IOSE523 and BT549.Overall, mAb C51 delivered saporin and induce potent cytotoxicity.Results demonstrated the use of mAb C51 as an antibody-drug conjugate(ADC).

mAb C51 induces complement-dependent cytotoxicity (CDC).

mAb C51 demonstrated detectable levels of cell lysis in the presence ofhuman complement (FIG. 7). Up to 30% lysis was observed at high bindingsaturation concentrations of C51. For control, cells were incubated withmAb and heat-inactivated complement. Results showed that mAb C51 can beused to induce CDC activity on target cancer cells.

Chimeric mAb chC51 as an antibody-drug conjugate (ADC) kills cancercells.

Ovarian and breast cancer cells were incubated in culture with chimericC51 mAb (chC51) and secondary anti-human conjugated to saporin (HZAP).After 72 h of incubation, relative cell viability was measured by thepercentage of live cells in the treatment group compared to thebuffer-treated control (FIG. 8). Overall, chimeric mAb chC51 deliveredsaporin and induce potent cytotoxicity. Results demonstrated the use ofchimeric mAb chC51 as an antibody-drug conjugate (ADC) comparable to themouse C51 IgM antibody.

mAb chC51 induces antibody-dependent cell-mediated cytotoxicity (ADCC).

The chimeric mAb chC51 induced ADCC against MCF7 breast cancer cells(FIG. 9). Positive ADCC activity corresponded to binding of chC51 oncells which was not observed on non-binding cancer cell lines.

Ab heavy and light chain sequences and isotype of mAb 2448 isIgG1-kappa.

Heavy and light chain gene sequences of mAb 2448 were cloned fromhybridoma cells by reverse transcription polymerase chain reaction(RT-PCR) and sequenced with complementarity-determining regions (CDRs)underlined (FIGS. 10A and B). Isotyping of supernatant from hybridomaclone supernatant revealed that mAb 2448 was an IgG1-kappaimmunoglobulin (FIG. 10C).

mAb 2448 demonstrates reactivity to various cancers. mAb 2448 binds toan epithelial phenotype according to a classification based on theEpithelial-Mesenchymal Transition (EMT).

High-throughput screening of mAb 2448 was done on live cells by flowcytometry (FIG. 11A). Cell surface binding was observed on breast(BT474, BT20, CAMA1, HCC1937, HCC1954, HCC2218, MS578T, MCF7, MDAMB453,SKBR3, T47D), colorectal (COLO205, HT29, KM12, LS174T, SW620), kidney(786O, A498, ACHN, CAKI1, UO31, RXF393, TK10), liver (HEP3B, HEPG2,HUH7, PLC) and ovarian (CAOV3, CH1, IGROV1, OV17R, OV90, OVCA432,OVCAR3, OVCAR8, OVCA433, PEA1, SKOV3) cancer cells. Minimal binding wasobserved on normal cell lines (IOSE523, HEK293, HFF). Results showedthat mAb 2448 can specifically bind to multiple cancers. Binding of mAb2448 to ovarian and breast cancer cells was re-organized according to anEpithelial-Mesenchymal Transition (EMT) classification (FIG. 11B). mAb2448 was shown to preferentially bind to cells classified with anepithelial (E) and intermediate epithelial (IE) phenotype. Resultsshowed that mAb 2448 can potentially be used to monitor EMT. Isogenicbreast cancer cell lines MCF7-D10 and MCF7-2101 display epithelial andmesenchymal phenotypes of EMT, respectively. Significantly higher levelsof the epithelial marker E-Cadherin and lower levels of the mesenchymalmarker Vimentin are expressed in MCF7 cells versus MCF72101 cells,respectively. Microscopy images of 2448 on MCF-D10 and MCF7-2101isogenic cell lines (FIG. 11C). Phase contrast images revealed EMT-likemorphological changes. MCF7-D10 cells were more epithelial with cuboidalor “cobblestone-like” cells compared to MCF7-2101 cells which were moreisolated and elongated. Membranous binding of 2448 was observed onMCF7-D10 cells but not on the isogenic MCF7-2101 cells byimmunocytochemistry. mAb 2448 binds on live MCF7-D10 cells, but not onMCF7-2101 cells.

Annexin A2 is identified as the antigen target of mAb 2448 in an ovariancancer cell line.

Immunoprecipitation (IP) was carried out on IGROV1 ovarian cancer cellsto enrich for the antigen of mAb 2448. IP product was immunoblotted with2448 (FIG. 12A). Corresponding bands on a silver stained gel wereexcised and analyzed by liquid chromatography tandem mass spectrometry(LC-MS/MS) Results demonstrated Annexin A2 as the antigen target of mAb2448 (FIG. 12B). Peptide coverage spanned across the entire amino acidsequence of ANXA2 after multiple rounds of IP and LC-MS/MS.

Annexin A2 is identified as the antigen target of mAb 2448 in hESC and abreast cancer cell line.

Immunoprecipitation (IP) was carried out on hESC and T47D breast cancercells to enrich for the antigen of mAb 2448. IP product wasimmunoblotted with mAb 2448 (FIG. 13A). Corresponding bands on a silverstained gel were excised and analyzed by liquid chromatography tandemmass spectrometry (LC-MS/MS). Annexin A2 was identified as a top proteinhit (FIG. 13B). Results demonstrated Annexin A2 as the antigen target ofmAb 2448.

Annexin A2 is validated as the antigen target of mAb 2448 in humanembryonic stem cells (hESC) and a breast cancer cell line.

To validate ANXA2 as the antigen target of mAb 2448, a forward andreverse-immunoprecipitation (IP) was carried out using a commercialanti-ANXA2 mAb. IP products were immunoblotted against mAb 2448 and thecommercial antibody (FIG. 14A). mAb 2448 recognized similar antigenbands for both IP products. A transient siRNA knockdown study of ANXA2was also carried out (FIG. 14B). Partial knockdown of ANXA2 correspondedto a loss of antigen recognition by mAb 2448. Taken together, resultsdemonstrated Annexin A2 as the antigen target of mAb 2448.

Only mAb 2448 binds to cell surface Annexin A2.

mAb 2448 was evaluated for binding on cancer cells. Strong cell surfacebinding was demonstrated for mAb 2448 unlike commercial anti-ANXA2antibodies (FIG. 15). Commercial antibodies only demonstrated bindingafter cells were fixed and permeabilized. Results demonstrate that mAb2448 binds to unique surface epitopes on cancer cells unlike otheranti-ANXA2 mAbs.

mAb 2448 targets an N-glycan epitope.

mAb 2448 binding was loss after periodate treatment (FIG. 16A) andPNGase F enzymatic release (FIG. 16B). Loss of binding demonstrated thatmAb 2448 was binding to N-glycan-dependent epitopes on ANXA2.

mAb 2448 internalizes into cancer cells.

mAb 2448 was conjugated to CypHER5E (FIG. 17A) and pHRodo dye (FIG.17B). Results demonstrated the ability of mAb 2448 to efficientlyinternalize into target cancer cells.

mAb 2448 and chimeric mAb ch2448 kill target cancer cells as antibodydrug conjugates (ADCs).

To demonstrate either 2448 or ch2448 as a potential ADC, mAbs wereevaluated with secondary conjugates of the plant-derived toxin, saporin.Ovarian and breast cancer cells were incubated with primary mAb (2448 orch2448), secondary saporin conjugate (mAb-ZAP or HUM-ZAP) or complexesof primary mAb and secondary conjugates (FIG. 18).

The complexes of primary mAb and secondary conjugates delivered saporininto cells and induced cytotoxicity. Overall, results indicated thatboth 2448 and ch2448 were viable targeting agents for development as anADC.

Chimeric mAb ch2448 as an antibody-drug conjugate (ADC) kills targetcancer cells in a dose-dependent manner.

Chimeric mAb 2448 was conjugated to a plant-derived toxin (saporin) tocreate an antibody drug conjugate (ADC). As an ADC, ch2448 killed IGROV1target ovarian cancer cells but not IOSE523 normal ovarian cells (FIG.19A). Dose dependent cytotoxicity was observed on IGROV1 and SKOV3ovarian cancer cell lines (FIG. 19B). Results demonstrated that chimericmAb ch2448 can be used as an ADC with potent cytotoxicity.

Chimeric mAb ch2448 induces antibody-dependent cell-mediatedcytotoxicity (ADCC).

Chimeric mAb ch2448 was co-incubated with target cancer cell lines andeffector cells. Dose dependent activation of the ADCC NFAT pathway wasmeasured by luciferase readout. Chimeric mAb ch2448 can be used toactivate ADCC activity against ovarian cancer cells (FIG. 20A) andbreast cancer cells (FIG. 20B).

Chimeric mAb 2448 delays tumor growth in vivo.

Chimeric mAb ch2448 delayed tumor growth in an IGROV1 ovarian cancerxenograft mouse model (FIG. 21A). No drastic changes in body weight wereobserved (FIG. 21B), suggesting no adverse effects of antibodytreatment. Results suggest that ch2448 can be used as a naked antibodytreatment for ovarian cancer.

Chimeric mAb ch2448 delays tumor growth in vivo via ADCC.

A F(ab)′2 of ch2448 was generated (FIG. 22A) with no ability to induceantibody-dependent cell-mediated cytotoxicity (ADCC) (FIG. 22B).Chimeric mAb ch2448 as an IgG but not as a F(ab′)2 delayed human ovariantumor growth in a mouse xenograft model (FIG. 22C). Results demonstratedthe ability of ch2448 to delay tumor growth via ADCC.

Afucosylation enhances the ability of ch2448 to induceantibody-dependent cell-mediated cytotoxicity (ADCC).

Afucosylated ch2448 retained similar binding to wildtype chimeric mAbch2448 (FIG. 23A). mAb ch2448 and aF-ch2448 were incubated with targetcancer cells and effector cells. aF-ch2448 induced greater ADCC activitycompared to ch2448 as indicated by an increase in fold change (FIG.23B). Results showed that afucosylation of ch2448 retainedantibody-antigen specificity and significantly enhanced ADCC activity ofch2448.

mAb ch2448 binds strongly to undifferentiated hESC and prevents teratomaformation in vivo.

mAb 2448 binds specifically to undifferentiated hESC but not todifferentiated embryoid bodies (EBs) (FIG. 24A). hESC were eitherpre-incubated with PBS buffer or with mAb 2448 prior to injecting intoSCID mice intramuscularly (FIG. 24B). Teratomas formed within week 5 inall 3 mice in the buffer control. Cells treated with 2448 saw preventionin teratoma (n=2) or a delay in teratoma (n=1, week 7). Hence, as anaked mAb, 2448 can prevent or delay the formation of teratoma in SCIDmice.

ch2448 internalizes into hESC and kills undifferentiated hESC as an ADC.

ch2448 was conjugated to pHRodo dye and internalization of ch2448 wasobserved over 24 hrs (FIG. 25A). hESC was partially differentiatedspontaneously and actin stained with phalloidin. Undifferentiated hESCshowed dense actin staining while differentiated cells had sparse actinstaining. ch2448 (conjugated to pHRodo) binds to and internalizes intoundifferentiated regions (FIG. 25B). ch2448 as a F(ab′)2 can beconjugated to a toxin and potentially be used as an ADC to killundifferentiated hESC. Hum-Zap was incubated with ch2448 as an ADCcomplex and spiked into undifferentiated hESC cultures. The ch2448-ADCkilled the undifferentiated hESC within 3 days (FIG. 25C). No killingwas observed for the buffer control, ch2448 alone and the isotypecontrol ADC. The ch2448 ADC does not kill differentiated embryoidbodies. The Fc region of ch2448 was removed enzymatically and complexedwith anti-Fab saporin as an ADC (FIG. 25D). The F(ab′)2-ADC killed theundifferentiated hESC within 3 days. No killing was observed for thebuffer control, F(ab)2 alone and the free saporin control.

mAb 2448 as a F(ab′)2-ADC prevents or delays teratoma formation in vivo.

Single-cell suspension of human embryonic stem cells (5×10⁶ cells peranimal) were pre-incubated with either buffer or mAb 2448 a F(ab′)2-ADCat 4° C. for 20 minutes and then injected into the right hind leg muscleof SCID mice. The F(ab′)2-ADC was able to prevent tumor formationcompared to the control group (FIG. 26A). Single-cell suspension ofhuman embryonic stem cells (5×10⁶ cells per animal) were injected intothe right hind leg muscle of SCID mice (n=3). Buffer and ADC weresubsequently administered intra-peritoneal.

Prevention or delayed teratoma formation by 2448-F(ab′)2-ADC in SCIDmouse was observed compared to the control group (FIG. 26B). Resultsdemonstrated the ability of mAb 2448 to be used as an ADC to prevent ordelay teratoma formation in vivo.

CAR(2448) was constructed using the CD28 co-stimulatory domain, and anIgG₄ Fc region linker domain. A T2A element and eGFP were inserteddownstream in the same open reading frame as the CAR construct.

A second generation CAR utilising the V_(H) and V_(L) regions of mAb2448 was constructed to determine the utility of the antibody in a CARformat. An eGFP element was utilised as a reporter for CAR expression(FIG. 27).

Key cytokines associated with T cell activation are upregulated uponco-incubation of CAR(2448) T cells with complement antigen-expressingtarget cells.

A significant increase in T cell cytokines by CAR(2448) T cells wasobserved upon exposure to IGROV-1 cells that express the target AnnexinA2 (FIG. 28). High levels of IFN-γ, IL-2, GM-CSF, and TNF-a suggestedthe activation of T cell subsets necessary for tumor clearance.

After T cells were co-incubated with target IGROV1 cells, multiplecytokines show a significant increase in cytokine production compared toa non-target specific CAR control.

The upregulation of multiple cytokines (GM-CSF, IFN-γ, IL-2, IL-4, IL-5,IL-6, IL-10, IL-17A, and TNF-α) by CAR(2448) T cells incubated withIGROV-1 suggested that multiple T cell subsets were activated,indicating the capability of the CAR(2448) construct in activating abroad range of T cell subsets (FIG. 29).

CAR(2448) mediates T cell cytotoxicity against target cells compared tonon-specific CAR control.

CAR(2448) T cells mediated cytotoxicity against target IGROV-1 cells ina dose-dependent manner (FIG. 30).

CAR(2448) T cells mediates growth inhibition of target cells.

Target IGROV-1 cells were co-incubated with CAR-T cells. All dose levelsof CAR(2448) T cells showed inhibition of target cell growth compared toa non-target specific CAR control (FIG. 31).

CAR(2448) T cells mediated their cytotoxic effect on IGROV1 target cellsover the course of several hours.

1.-62. (canceled)
 63. An antigen-binding protein, or an antigen-bindingfragment thereof, comprising: (a)(i) a heavy chain variable domaincomprising a VHCDR1 having the amino acid sequence GYSITSGYSWH (SEQ IDNO: 9); a VHCDR2 having the amino acid sequence YIHYSGSTKYNPSLKS (SEQ IDNO: 10) and a VHCDR3 having the amino acid sequence GSNYGFDY (SEQ NO:11); and (ii) a light chain variable domain comprising a VLCDR1 havingthe amino acid sequence KSSQSLLYSNDQKNYLA (SEQ ID NO: 12), a VLCDR2having the amino acid sequence WASIRES (SEQ ID NO: 13), and a VLCDR3having the amino acid sequence QQYYIYPLT (SEQ ID NO: 14), or (b) (i) aheavy chain variable domain comprising a VHCDR1 having the amino acidsequence VYSITSGYSWH (SEQ ID NO: 21); a VHCDR2 having the amino acidsequence YIHYSGSTKYNPSLKS (SEQ ID NO: 10), and a VHCDR3 having the aminoacid sequence GTDNAVDY (SEQ ID NO: 22); and (ii) a light chain variabledomain comprising a VLCDR1 having the amino acid sequenceKSSQSLLYSSNQKNYLA (SEQ ID NO: 23), a VLCDR2 having the amino acidsequence WASSRES (SEQ ID NO: 24), and a VLCDR3 having the amino acidsequence QQYYIYPLT (SEQ ID NO: 14).
 64. The antigen-binding protein, orantigen-binding fragment thereof, as claimed in claim 63, comprisingheavy and light chain CDR regions that are about 80%, about 85%, about90%, about 95%, about 96%, about 97%, about 98% or about 99% identicalto the heavy and light chain CDR regions of (i) and (ii).
 65. Theantigen-binding protein, or antigen-binding fragment thereof, as claimedin claim 63, wherein the heavy chain variable region of (a) comprisesthe amino acid sequence set forth in SEQ ID NO: 1, or wherein the heavychain variable region of (b) comprises the amino acid sequence set forthin SEQ ID NO:5; optionally wherein the antigen-binding protein, orantigen-binding fragment thereof comprises a heavy chain variable regionwhich comprises an amino acid sequence having about 80%, about 85%,about 90%, about 95%, about 96%, about 97%, about 98% or about 99%identity to the amino acid sequence set forth in SEQ ID NO: I or SEQ IDNO:
 5. 66. The antigen-binding protein, or antigen-binding fragmentthereof, as claimed in claim 63, wherein the light chain variable regionof (a) comprises the amino acid sequence set forth in SEQ ID NO:2, orwherein the light chain variable region of (b) comprises the amino acidsequence set forth in SEQ ID NO: 6; optionally wherein the light chainvariable region comprises an amino acid sequence having about 80%, about85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99%identity to the amino acid sequence set forth in SEQ ID NO:2 or SEQ IDNO:
 6. 67. An antigen-binding protein, or an antigen-binding fragmentthereof, comprising: (a) (i) a heavy chain variable domain comprising aVHCDR1 having the nucleic acid sequence GCTACTCCATCACCAGTGGTTATAGCTGGCAC(SEQ ID NO: 15); a VHCDR2 having the nucleic acid sequenceACATACACTACAGTGGTAGCACTAAGTACAACCCATCTCTCAAAAGTC (SEQ ID NO: 16), and aVHCDR3 having the nucleic acid sequence GGAGTAACTACGGATTTGACTACT (SEQ IDNO: 17); and (ii) a light chain variable domain comprising a VLCDR1having the nucleic acid sequenceAGTCCAGTCAGAGCCTTTTATATAGTAACGATCAAAAGAACTACTTGGCC T (SEQ ID NO: 18), aVLCDR2 having the nucleic acid sequence GGGCATCTATTAGGGAATCFG (SEQ IDNO: 19), and a VLCDR3 having the nucleic acid sequenceAGCAATATTATATCTATCCTCTCACGT (SEQ ID NO: 20), or (b) (i) a heavy chainvariable domain comprising a VHCDR1 having the nucleic acid sequenceTCTACTCCATCACCAGTGGTTATAGCTGGCACT (SEQ ID NO: 25); a VHCDR2 having thenucleic acid sequence ACATACACTACAGTGGTAGTACTAAGTACAACCCATCTCTCAAAAGTC(SEQ ID NO: 26), and a VHCDR3 having the nucleic acid sequenceGGACCGACAATGCTGTGACTACT (SEQ ID NO: 27); and (ii) a light chain variabledomain comprising a VLCDR1 having the nucleic acid sequenceAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCC T (SEQ ID NO: 28), aVLCDR2 having the nucleic acid sequence GGGCATCCAGTAGGGAATCTG (SEQ IDNO: 29), and a VLCDR3 having the nucleic acid sequenceAGCAATATFATATCTATCCTCTCACGT (SEQ ID NO: 20).
 68. The antigen-bindingprotein, or antigen-binding fragment thereof, as claimed in claim 67,comprising heavy and light chain CDR regions that are about 60%, 65%,70%, 75%, 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98% or about 99% identical to the heavy and light chain CDRregions of (i) and (ii).
 69. The antigen-binding protein, orantigen-binding fragment thereof, as claimed in claim 67, wherein theheavy chain variable region of (a) comprises the nucleic acid sequenceset forth in SEQ ID NO: 3, or wherein the heavy chain variable region of(b) comprises the nucleic acid sequence set forth in SEQ NO:7;optionally wherein the heavy chain variable region comprises a nucleicacid sequence having about 60%, 65%, 70%, 75%, 80%, about 85%, about90%, about 95%, about 96%, about 97%, about 98% or about 99% identity tothe nucleic acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:
 7. 70.The antigen-binding protein, or antigen-binding fragment thereof, asclaimed in claim 67, wherein the light chain variable region of (a)comprises the nucleic acid sequence set forth in SEQ ID NO:4, or whereinthe light chain variable region of (b) comprises the nucleic acidsequence set forth in SEQ ID NO: 8; optionally wherein the light chainvariable region comprises a nucleic acid sequence having about 60%, 65%,70%, 75%, 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98% or about 99% identity to the amino acid sequence set forth inSEQ ID NO:4 or SEQ ID NO: 8,
 71. The antigen-binding protein, orantigen-binding fragment thereof, as claimed in claim 63, wherein theantigen binding protein is selected from the group consisting ofmonoclonal, recombinant, polyclonal, chimeric, humanised, bispecific andheteroconjugate antibodies; a chimeric antigen receptor (CAR), a singlevariable domain, a domain antibody, antigen binding fragments,immunologically effective fragments, single chain Fv, a single chainantibody, a univalent antibody lacking a hinge region, a minibody,diabodies, and Tandabs™; optionally wherein the binding protein is amonoclonal antibody; optionally wherein the monoclonal antibody is 2448or C51; optionally wherein the monoclonal antibody is humanized;optionally wherein the monoclonal antibody is chimeric; optionallywherein the monoclonal antibody is defucosylated; optionally wherein thedegree of fucosylation is less than 10%, preferably less than 50 andmore preferably is less than 1.5% relative to the wild-type antibody.72. The antigen-binding protein, or antigen-binding fragment thereof, asclaimed in claim 63, wherein the antigen-binding protein, orantigen-binding fragment thereof, binds to ANXA2; optionally wherein theantigen-binding protein, or antigen-binding fragment thereof binds to aglycan on ANXA2; optionally wherein the antigen-binding protein, or anantigen-binding fragment thereof binds to an N-linked glycan or a ANXA2;optionally wherein the linked glycan is located at amino acid residue 62of ANXA2.
 73. The antigen-binding protein, or antigen-binding fragmentthereof, as claimed in claim 63, further comprising a radioisotope or acytotoxin conjugated thereto; optionally wherein the antibody isconjugated with a cytotoxin selected from the group consisting ofmonomethyl auristatin E (MME-1), mertansine (DM-1) and saporin;optionally wherein the antigen-binding protein, or an antigen-bindingfragment is internalized into a cell upon binding to ANXA2; optionallywherein the antigen-binding protein, or an antigen-binding fragmentthereof has a cytotoxic activity selected from one or more of the groupconsisting of complement dependent cytotoxic (CDC) activity, antibodydependent cellular cytotoxic (ADCC) activity and oncolytic activity. 74.A composition comprising a physiologically acceptable carrier and atherapeutically effective amount of an antigen-binding protein, or anantigen-binding fragment thereof comprising: (a)(i) a heavy chainvariable domain comprising a VHCDR1 having the amino acid sequenceGYSITSGYSWH (SEQ ID NO: 9); a VHCDR2 having the amino acid sequenceYIHYSGSTKYNPSLKS (SEQ ID NO: 10) and a VHCDR3 having the amino acidsequence GSNYGFDY (SEQ ID NO: 11); and (ii) a light chain variabledomain comprising VLCDR1 having the amino acid sequenceKSSQSLLYSNDQKNYLA (SEQ ID NO: 12), a VLCDR2 having the amino acidsequence WASIRES (SEQ ID NO: 13), and a VLCDR3 having the amino acidsequence QQYYIYPLT (SEQ ID NO: 14), or (b) (i) a heavy chain variabledomain comprising a VHCDR1 having the amino acid sequence VYSITSGYSWH(SEQ ID NO: 21); a VHCDR2 having the amino acid sequenceYIHYSGSTKYNPSLKS (SEQ ID NO: 10), and a VHCDR3 having the amino acidsequence GTDNAVDY (SEQ ID NO: 22); and (ii) a light chain variabledomain comprising a VLCDR1 having the amino acid sequenceKSSQSLLYSSNQKNYLA (SEQ ID NO: 23), a VLCDR2 having the amino acidsequence WASSRES (SEQ ID NO: 24), and a VLCDR3 having the amino acidsequence QQYYTYPLT (SEQ ID NO: 14).
 75. The composition as claimed inclaim 74, wherein the composition comprises a further activepharmaceutical ingredient selected from the group consisting ofbevacizumab, carboplatin, paclitaxel, or gefitinib.